RESUMO
L-type amino acid transporter 1 (LAT1) is recognized as a promising target for cancer therapy; however, the cellular adaptive response to its pharmacological inhibition remains largely unexplored. This study examined the adaptive response to LAT1 inhibition using nanvuranlat, a high-affinity LAT1 inhibitor. Proteomic analysis revealed the activation of a stress-induced transcription factor ATF4 following LAT1 inhibition, aligning with the known cellular responses to amino acid deprivation. This activation was linked to the GCN2-eIF2α pathway which regulates translation initiation. Our results show that ATF4 upregulation counteracts the suppressive effect of nanvuranlat on cell proliferation in pancreatic ductal adenocarcinoma cell lines, suggesting a role for ATF4 in cellular adaptation to LAT1 inhibition. Importantly, dual targeting of LAT1 and ATF4 exhibited more substantial anti-proliferative effects in vitro than individual treatments. This study underscores the potential of combining LAT1 and ATF4 inhibition as a therapeutic strategy in cancer treatment.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Regulação para Cima , Proteômica , Aminoácidos/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Carcinoma Ductal Pancreático/tratamento farmacológico , Transportador 1 de Aminoácidos Neutros Grandes/genética , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Linhagem Celular Tumoral , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismoRESUMO
Amino acid transporter LAT1 is highly upregulated in various cancer types, including cholangiocarcinoma (CHOL), and contributes to the rapid proliferation of cancer cells and disease progression. However, the molecular mechanisms underlying the pathological upregulation of LAT1 remain largely unknown. This study pursued the possibility of miRNA-mediated regulation of the LAT1 expression in CHOL cells. Using online target prediction methods, we extracted five candidate miRNAs commonly predicted to regulate the LAT1 expression. Three of them, miR-194-5p, miR-122-5p, and miR-126-3p, were significantly downregulated in CHOL cancer compared to normal tissues. Correlation analysis revealed weak-to-moderate negative correlations between the expression of these miRNAs and LAT1 mRNA in CHOL cancer tissues. We selected miR-194-5p and miR-122-5p for further analyses and found that both miRNAs functionally target 3'UTR of LAT1 mRNA by a luciferase-based reporter assay. Transfection of the miRNA mimics significantly suppressed the LAT1 expression at mRNA and protein levels and inhibited the proliferation of CHOL cells, with a trend of affecting intracellular amino acids and amino acid-related signaling pathways. This study indicates that the decreased expression of these LAT1-targeting tumor-suppressive miRNAs contributes to the upregulation of LAT1 and the proliferation of CHOL cells, highlighting their potential for developing novel cancer therapeutics and diagnostics.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , MicroRNAs , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Linhagem Celular Tumoral , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , RNA Mensageiro/genética , Regulação Neoplásica da Expressão Gênica , Proliferação de Células/genéticaRESUMO
Scavenger receptors are a protein superfamily that typically consists of one or more repeats of the scavenger receptor cysteine-rich structural domain (SRCRD), which is an ancient and highly conserved protein module. The expression and purification of eukaryotic proteins containing multiple disulfide bonds has always been challenging. The expression systems that are commonly used to express SRCRD proteins mainly consist of eukaryotic protein expression systems. Herein, we established a high-level expression strategy of a Type B SRCRD unit from human salivary agglutinin using the Escherichia coli expression system, followed by a refolding and purification process. The untagged recombinant SRCRD was expressed in E. coli using the pET-32a vector, which was followed by a refolding process using the GSH/GSSG redox system. The SRCRD expressed in E. coli SHuffle T7 showed better solubility after refolding than that expressed in E. coli BL21(DE3), suggesting the importance of the disulfide bond content prior to refolding. The quality of the refolded protein was finally assessed using crystallization and crystal structure analysis. As proteins refolded from inclusion bodies exhibit a high crystal quality and reproducibility, this method is considered a reliable strategy for SRCRD protein expression and purification. To further confirm the structural integrity of the refolded SRCRD protein, the purified protein was subjected to crystallization using sitting-drop vapor diffusion method. The obtained crystals of SRCRD diffracted X-rays to a resolution of 1.47 Å. The solved crystal structure appeared to be highly conserved, with four disulfide bonds appropriately formed. The surface charge distribution of homologous SRCRD proteins indicates that the negatively charged region at the surface is associated with their calcium-dependent ligand recognition. These results suggest that a high-quality SRCRD protein expressed by E. coli SHuffle T7 can be successfully folded and purified, providing new options for the expression of members of the scavenger receptor superfamily.
Assuntos
Escherichia coli , Redobramento de Proteína , Proteínas Recombinantes , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Cristalografia por Raios X , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Cristalização , Aglutininas/química , Aglutininas/genética , Aglutininas/metabolismo , Domínios Proteicos , Expressão Gênica , Modelos Moleculares , Cisteína/química , Cisteína/genética , Receptores Depuradores/química , Receptores Depuradores/genética , Receptores Depuradores/metabolismoRESUMO
L-type amino acid transporter 1 (LAT1; SLC7A5), which preferentially transports large neutral amino acids, is highly upregulated in various cancers. LAT1 supplies cancer cells with amino acids as substrates for enhanced biosynthetic and bioenergetic reactions and stimulates signalling networks involved in the regulation of survival, growth and proliferation. LAT1 inhibitors show anti-cancer effects and a representative compound, JPH203, is under clinical evaluation. However, pharmacological impacts of LAT1 inhibition on the cellular amino acid transport and the translational activity in cancer cells that are conceptually pivotal for its anti-proliferative effect have not been elucidated yet. Here, we demonstrated that JPH203 drastically inhibits the transport of all the large neutral amino acids in pancreatic ductal adenocarcinoma cells. The inhibitory effects of JPH203 were observed even in competition with high concentrations of amino acids in a cell culture medium. The analyses of the nutrient-sensing mTORC1 and GAAC pathways and the protein synthesis activity revealed that JPH203 downregulates the global translation. This study demonstrates a predominant contribution of LAT1 to the transport of large neutral amino acids in cancer cells and the suppression of protein synthesis by JPH203 supposed to underly its broad anti-proliferative effects across various types of cancer cells.
Assuntos
Aminoácidos Neutros , Neoplasias , Aminoácidos , Linhagem Celular Tumoral , Transportador 1 de Aminoácidos Neutros Grandes/genética , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismoRESUMO
Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states at 3.2 Å and 2.8 Å, respectively. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. We provide the characterization of the molecular features of EAAT2 and its selective inhibition mechanism that may facilitate structure-based drug design for EAAT2.
Assuntos
Transportador 2 de Aminoácido Excitatório/química , Ácido Glutâmico , Animais , Sítios de Ligação , Encéfalo/metabolismo , Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Transportador 3 de Aminoácido Excitatório/genética , Transportador 3 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Mamíferos/metabolismo , Neuroglia/metabolismoRESUMO
BACKGROUND: New strategies are needed to combat multidrug-resistant bacteria. The restriction of iron uptake by bacteria is a promising way to inhibit their growth. We aimed to suppress the growth of Vibrio bacterial species by inhibiting their ferric ion-binding protein (FbpA) using food components. METHODS: Twenty spices were selected for the screening of FbpA inhibitors. The candidate was applied to antibacterial tests, and the mechanism was further studied. RESULTS: An active compound, rosmarinic acid (RA), was screened out. RA binds competitively and more tightly than Fe3+ to VmFbpA, the FbpA from V. metschnikovii, with apparent KD values of 8 µM vs. 17 µM. Moreover, RA can inhibit the growth of V. metschnikovii to one-third of the control at 1000 µM. Interestingly, sodium citrate (SC) enhances the growth inhibition effect of RA, although SC only does not inhibit the growth. The combination of RA/SC completely inhibits the growth of not only V. metschnikovii at 100/100 µM but also the vibriosis-causative pathogens V. vulnificus and V. parahaemolyticus, at 100/100 and 1000/100 µM, respectively. However, RA/SC does not affect the growth of Escherichia coli. CONCLUSIONS: RA/SC is a potential bacteriostatic agent against Vibrio species while causing little damage to indigenous gastrointestinal bacteria.
Assuntos
Cinamatos/farmacologia , Depsídeos/farmacologia , Ferro/metabolismo , Citrato de Sódio/farmacologia , Vibrio parahaemolyticus/efeitos dos fármacos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cinamatos/química , Cinamatos/metabolismo , Depsídeos/química , Depsídeos/metabolismo , Sinergismo Farmacológico , Proteínas de Ligação ao Ferro/química , Proteínas de Ligação ao Ferro/metabolismo , Simulação de Acoplamento Molecular , Extratos Vegetais/química , Ligação Proteica , Vibrio parahaemolyticus/metabolismo , Ácido RosmarínicoRESUMO
L-type amino acid transporter 1 (LAT1) is highly expressed in various cancers and plays important roles not only in the amino acid uptake necessary for cancer growth but also in cellular signaling. Recent research studies have reported anticancer effects of LAT1 inhibitors and demonstrated their potential for cancer therapy. Here, we characterized the proteome and phosphoproteome in LAT1-inhibited cancer cells. We used JPH203, a selective LAT1 inhibitor, and performed tandem mass tag-based quantitative proteomics and phosphoproteomics on four biliary tract cancer cell lines sensitive to JPH203. Our analysis identified hundreds to thousands of differentially expressed proteins and phosphorylated sites, demonstrating the broad influence of LAT1 inhibition. Our findings showed various functional pathways altered by LAT1 inhibition, and provided possible regulators and key kinases in LAT1-inhibited cells. Comparison of these changes among cell lines provides insights into general pathways and regulators associated with LAT1 inhibition and particularly suggests the importance of cell cycle-related pathways and kinases. Moreover, we evaluated the anticancer effects of the combinations of JPH203 with cell cycle-related kinase inhibitors and demonstrated their potential for cancer therapy. This is the first study providing the proteome-wide scope of both protein expression and phosphorylation signaling perturbed by LAT1 inhibition in cancer cells.
Assuntos
Benzoxazóis/farmacologia , Proliferação de Células/efeitos dos fármacos , Citostáticos/farmacologia , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Tirosina/análogos & derivados , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Proteômica , Tirosina/farmacologiaRESUMO
BACKGROUND: Tumor angiogenesis is regarded as a rational anti-cancer target. The efficacy and indications of anti-angiogenic therapies in clinical practice, however, are relatively limited. Therefore, there still exists a demand for revealing the distinct characteristics of tumor endothelium that is crucial for the pathological angiogenesis. L-type amino acid transporter 1 (LAT1) is well known to be highly and broadly upregulated in tumor cells to support their growth and proliferation. In this study, we aimed to establish the upregulation of LAT1 as a novel general characteristic of tumor-associated endothelial cells as well, and to explore the functional relevance in tumor angiogenesis. METHODS: Expression of LAT1 in tumor-associated endothelial cells was immunohistologically investigated in human pancreatic ductal adenocarcinoma (PDA) and xenograft- and syngeneic mouse tumor models. The effects of pharmacological and genetic ablation of endothelial LAT1 were examined in aortic ring assay, Matrigel plug assay, and mouse tumor models. The effects of LAT1 inhibitors and gene knockdown on cell proliferation, regulation of translation, as well as on the VEGF-A-dependent angiogenic processes and intracellular signaling were investigated in in vitro by using human umbilical vein endothelial cells. RESULTS: LAT1 was highly expressed in vascular endothelial cells of human PDA but not in normal pancreas. Similarly, high endothelial LAT1 expression was observed in mouse tumor models. The angiogenesis in ex/in vivo assays was suppressed by abrogating the function or expression of LAT1. Tumor growth in mice was significantly impaired through the inhibition of angiogenesis by targeting endothelial LAT1. LAT1-mediated amino acid transport was fundamental to support endothelial cell proliferation and translation initiation in vitro. Furthermore, LAT1 was required for the VEGF-A-dependent migration, invasion, tube formation, and activation of mTORC1, suggesting a novel cross-talk between pro-angiogenic signaling and nutrient-sensing in endothelial cells. CONCLUSIONS: These results demonstrate that the endothelial LAT1 is a novel key player in tumor angiogenesis, which regulates proliferation, translation, and pro-angiogenic VEGF-A signaling. This study furthermore indicates a new insight into the dual functioning of LAT1 in tumor progression both in tumor cells and stromal endothelium. Therapeutic inhibition of LAT1 may offer an ideal option to potentiate anti-angiogenic therapies.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Carcinoma Ductal Pancreático/irrigação sanguínea , Endotélio Vascular/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Neoplasias Pancreáticas/irrigação sanguínea , Fator A de Crescimento do Endotélio Vascular/metabolismo , Sistema y+L de Transporte de Aminoácidos/metabolismo , Animais , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Endotélio Vascular/patologia , Feminino , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neoplasias Pancreáticas/metabolismo , Transdução de SinaisRESUMO
Halogenated tyrosine/phenylalanine derivatives have been developed for use in tumor imaging and targeted alpha therapy. 3-Fluoro-α-methyl-l-tyrosine (FAMT), targeting amino acid transporter LAT1 (SLC7A5), is a cancer-specific positron emission tomography probe that exhibits high renal accumulation, which is supposed to be mediated by organic anion transporter OAT1 (SLC22A6). In the present study, we investigated the structural requirements of FAMT essential for interaction with OAT1. OAT1 transported FAMT with a K m of 171.9 µM. In structure-activity relationship analyses, removal of either the 3-halogen or 4-hydroxyl group from FAMT or its structural analog 3-iodo-α-methyl-l-tyrosine greatly decreased the interaction with OAT1, reducing the [14C]p-aminohippurate uptake inhibition and the efflux induction. By contrast, the α-methyl group, which is essential for LAT1 specificity, contributed to a lesser degree. In fluorinated tyrosine derivatives, fluorine at any position was accepted by OAT1 when there was a hydroxyl group at the ortho-position, whereas ortho-fluorine was less interactive when a hydroxyl group was at meta- or para-positions. The replacement of the ortho-fluorine with a bulky iodine atom greatly increased the interaction. In in vivo studies, probenecid decreased the renal accumulation (P < 0.001) and urinary excretion (P = 0.0012) of FAMT, whereas the plasma concentration was increased, suggesting the involvement of OAT1-mediated transepithelial organic anion excretion. LAT1-specific 2-fluoro-α-methyltyrosine, which had lower affinity for OAT1, exhibited lower renal accumulation (P = 0.0142) and higher tumor uptake (P = 0.0192) compared with FAMT. These results would provide a basis to design tumor-specific compounds that can avoid renal accumulation for tumor imaging and targeted alpha therapy. SIGNIFICANCE STATEMENT: We revealed the structural characteristics of halogenated tyrosine derivatives essential for interaction with the organic anion transporter responsible for their renal accumulation. We have confirmed that such interactions are important for renal handling and tumor uptake. The critical contribution of hydroxyl and halogen groups and their positions as well as the role of α-methyl group found in the present study may facilitate the development of tumor-specific compounds while avoiding renal accumulation for use in tumor imaging and targeted alpha therapy.
Assuntos
Rim/diagnóstico por imagem , Metiltirosinas/metabolismo , Imagem Molecular/métodos , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Metiltirosinas/química , Metiltirosinas/farmacocinética , Camundongos , Ligação Proteica , Distribuição TecidualRESUMO
The L-type amino acid transporter 1 (LAT1 or SLC7A5) transports large neutral amino acids across the membrane and is crucial for brain drug delivery and tumor growth. LAT1 forms a disulfide-linked heterodimer with CD98 heavy chain (CD98hc, 4F2hc or SLC3A2), but the mechanism of assembly and amino acid transport are poorly understood. Here we report the cryo-EM structure of the human LAT1-CD98hc heterodimer at 3.3-Å resolution. LAT1 features a canonical Leu T-fold and exhibits an unusual loop structure on transmembrane helix 6, creating an extended cavity that might accommodate bulky amino acids and drugs. CD98hc engages with LAT1 through the extracellular, transmembrane and putative cholesterol-mediated interactions. We also show that two anti-CD98 antibodies recognize distinct, multiple epitopes on CD98hc but not its glycans, explaining their robust reactivities. These results reveal the principles of glycoprotein-solute carrier assembly and provide templates for improving preclinical drugs and antibodies targeting LAT1 or CD98hc.
Assuntos
Cadeia Pesada da Proteína-1 Reguladora de Fusão/química , Transportador 1 de Aminoácidos Neutros Grandes/química , Microscopia Crioeletrônica , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Cadeia Pesada da Proteína-1 Reguladora de Fusão/ultraestrutura , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/ultraestrutura , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Multimerização ProteicaRESUMO
Boron neutron capture therapy (BNCT) is a radiotherapy utilizing the neutron capture and nuclear fission reaction of 10B taken up into tumor cells. The most commonly used boron agent in BNCT, p-borono-l-phenylalanine (BPA), is accumulated in tumors by amino acid transporters upregulated in tumor cells. Here, by using dipeptides of BPA and tyrosine (BPA-Tyr and Tyr-BPA), we propose a novel strategy of selective boron delivery into tumor cells via oligopeptide transporter PEPT1 upregulated in various cancers. Kinetic analyses indicated that BPA-Tyr and Tyr-BPA are transported by oligopeptide transporters, PEPT1 and PEPT2. The intrinsic oligopeptide transport activity in tumor cells clearly correlated with PEPT1 protein expression level but not with PEPT2, suggesting that PEPT1 is the predominant oligopeptide transporter at least in tumor cell lines. Furthermore, using BPA-Tyr and Tyr-BPA, boron was successfully delivered into PEPT1-expressing pancreatic cancer AsPC-1 cells via a PEPT1-mediated mechanism. Intravenous administration of BPA-Tyr into the mice bearing AsPC-1 xenograft tumors resulted in significant boron accumulation in the tumors. It is proposed that the oligopeptide transporters, especially PEPT1, are promising candidates for molecular targets of boron delivery in BNCT. The BPA-containing dipeptides would have a potential for the development of novel boron carriers targeting PEPT1.
Assuntos
Compostos de Boro/administração & dosagem , Terapia por Captura de Nêutron de Boro/métodos , Neoplasias Pancreáticas/radioterapia , Transportador 1 de Peptídeos/genética , Fenilalanina/análogos & derivados , Animais , Transporte Biológico , Compostos de Boro/química , Compostos de Boro/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fenilalanina/administração & dosagem , Fenilalanina/química , Fenilalanina/metabolismo , Simportadores/genética , Tirosina/química , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Various physiological and pathological processes are accompanied with the alteration of pH at extracellular juxtamembrane region. Accordingly, the methods to analyze the cell surface pH have been demanded in biological and medical sciences. In this study, we have established a novel methodology for cell surface pH imaging using poly(ethylene glycol)-phospholipid (PEG-lipid) as a core structure of ratiometric fluorescent probes. PEG-lipid is a synthetic amphiphilic polymer originally developed for the cell surface modification in transplantation therapy. Via its hydrophobic alkyl chains of the phospholipid moiety, PEG-lipid is, when applied extracellularly, spontaneously inserted into the plasma membrane and retained at the surface of the cells. We have demonstrated that the PEG-lipid conjugated with fluorescein isothiocyanate (FITC-PEG-lipid) can be used as a sensitive and reversible cell-surface-anchored pH probe between weakly alkaline and acidic pH with an excellent spatiotemporal resolution. The remarkably simple procedure for cell-surface labeling with FITC-PEG-lipid would also be advantageous when considering its application to high-throughput in vitro assay. This study further indicates that various probes useful for the investigation of juxtamembrane environments could also be developed by using PEG-lipid as the core structure for bio-membrane anchoring.
Assuntos
Membrana Celular/metabolismo , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes , Imagem Óptica , Fosfolipídeos/química , Polietilenoglicóis/química , Linhagem Celular Tumoral , Humanos , Concentração de Íons de Hidrogênio , Microscopia Confocal , Soluções , Espectrometria de FluorescênciaRESUMO
Novobiocin is an orally active antibiotic that inhibits DNA gyrase by binding the ATP-binding site in the ATPase subunit. Although effective against Gram-positive pathogens, novobiocin has limited activity against Gram-negative organisms due to the presence of the lipopolysaccharide-containing outer membrane, which acts as a permeability barrier. Using a novobiocin-sensitive Escherichia coli strain with a leaky outer membrane, we identified a mutant with increased resistance to novobiocin. Unexpectedly, the mutation that increases novobiocin resistance was not found to alter gyrase, but the ATPase that powers lipopolysaccharide (LPS) transport. Co-crystal structures, biochemical, and genetic evidence show novobiocin directly binds this ATPase. Novobiocin does not bind the ATP binding site but rather the interface between the ATPase subunits and the transmembrane subunits of the LPS transporter. This interaction increases the activity of the LPS transporter, which in turn alters the permeability of the outer membrane. We propose that novobiocin will be a useful tool for understanding how ATP hydrolysis is coupled to LPS transport.
Assuntos
Adenosina Trifosfatases/metabolismo , Antibacterianos/metabolismo , Lipopolissacarídeos/metabolismo , Novobiocina/metabolismo , Novobiocina/farmacologia , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Transporte Biológico/efeitos dos fármacos , DNA Girase/metabolismo , Ativação Enzimática/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Hidrólise/efeitos dos fármacosRESUMO
L-type amino acid transporter 1 (LAT1) is known as a cancer-type amino acid transporter. In cancer cells, LAT1 is responsible for the cellular uptake of many essential amino acids including leucine that activates mechanistic/mammalian target of rapamycin (mTOR), regulating cancer cell growth. In this study, we designed a novel series of LAT1 inhibitors, SKN101-105, based on the structure of triiodothyronine (T3), a known LAT1 blocker. The compounds consist of core structure of 2-amino-3-[3,5-dichloro-4-(naphthalene-1-methoxy)-phenyl]-propanoic acid and different modifications on the naphthalene. Among them, the compounds including SKN103 with a modified phenyl group at C-7 position of naphthalene inhibited LAT1-mediated leucine transport, whereas SKN102 with a phenyl group at C-6 position did not, indicating the importance of the position of substituents on the naphthalene for the interaction with LAT1. SKN103 was suggested to be a non-transportable blocker rather than a substrate of LAT1 and inhibited LAT1 in a competitive manner with the Ki value of 2.1 µM. SKN103 suppressed mTOR activity and the growth of cancer cells. Moreover, SKN103 in combination with cisplatin additively enhanced the growth inhibition in cancer cells. This study provides an additional insight into the structure-activity relationship of LAT1 ligands, which could lead to designing desirable LAT1 inhibitors.
Assuntos
Transportador 1 de Aminoácidos Neutros Grandes/química , Tri-Iodotironina/análogos & derivados , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Cisplatino/farmacologia , Humanos , Leucina/metabolismo , Estrutura Molecular , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/metabolismo , Tri-Iodotironina/químicaRESUMO
A PET tracer for tumor imaging, 3-(18)F-l-α-methyl-tyrosine ([(18)F]FAMT), has advantages of high cancer-specificity and low physiological background. In clinical studies, FAMT-PET has been proved useful for the detection of malignant tumors and their differentiation from inflammation and benign lesions. The tumor specific uptake of FAMT is due to its high-selectivity to cancer-type amino acid transporter LAT1 among amino acid transporters. In [(18)F]FAMT PET, kidney is the only organ that shows high physiological background. To reveal transporters involved in renal accumulation of FAMT, we have examined [(14)C]FAMT uptake on the organic ion transporters responsible for the uptake into tubular epithelial cells. We have found that OAT1, OAT10 and OCTN2 transport [(14)C]FAMT. The [(14)C]FAMT uptake was inhibited by probenecid, furosemide and ethacrynic acid, consistent with the properties of the transporters. The amino acid uptake inhibitor, 2-amino-2-norbornanecarboxylic acid (BCH), also inhibited the [(14)C]FAMT uptake, whereas OCTN2-mediated [(14)C]FAMT uptake was Na(+)-dependent. We propose that FAMT uptake by OAT1, OAT10 and OCTN2 into tubular epithelial cells could contribute to the renal accumulation of FAMT. The results from this study would provide clues to the treatments to reduce renal background and enhance tumor uptake as well as to designing PET tracers with less renal accumulation.
Assuntos
Radioisótopos de Flúor , Rim/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Metiltirosinas , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos , Animais , Transporte Biológico , Células Cultivadas , Células Epiteliais/metabolismo , Radioisótopos de Flúor/metabolismo , Humanos , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Metiltirosinas/metabolismo , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismo , Oócitos/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Compostos Radiofarmacêuticos/metabolismo , Membro 5 da Família 22 de Carreadores de Soluto , Xenopus laevisRESUMO
Heterodimeric amino acid transporters play crucial roles in epithelial transport, as well as in cellular nutrition. Among them, the heterodimer of a membrane protein b(0,+)AT/SLC7A9 and its auxiliary subunit rBAT/SLC3A1 is responsible for cystine reabsorption in renal proximal tubules. The mutations in either subunit cause cystinuria, an inherited amino aciduria with impaired renal reabsorption of cystine and dibasic amino acids. However, an unsolved paradox is that rBAT is highly expressed in the S3 segment, the late proximal tubules, whereas b(0,+)AT expression is highest in the S1 segment, the early proximal tubules, so that the presence of an unknown partner of rBAT in the S3 segment has been proposed. In this study, by means of coimmunoprecipitation followed by mass spectrometry, we have found that a membrane protein AGT1/SLC7A13 is the second partner of rBAT. AGT1 is localized in the apical membrane of the S3 segment, where it forms a heterodimer with rBAT. Depletion of rBAT in mice eliminates the expression of AGT1 in the renal apical membrane. We have reconstituted the purified AGT1-rBAT heterodimer into proteoliposomes and showed that AGT1 transports cystine, aspartate, and glutamate. In the apical membrane of the S3 segment, AGT1 is suggested to locate itself in close proximity to sodium-dependent acidic amino acid transporter EAAC1 for efficient functional coupling. EAAC1 is proposed to take up aspartate and glutamate released into luminal fluid by AGT1 due to its countertransport so that preventing the urinary loss of aspartate and glutamate. Taken all together, AGT1 is the long-postulated second cystine transporter in the S3 segment of proximal tubules and a possible candidate to be involved in isolated cystinuria.
Assuntos
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Membrana Celular/metabolismo , Cistinúria/metabolismo , Túbulos Renais Proximais/metabolismo , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/genética , Animais , Anticorpos/metabolismo , Western Blotting , Transportador 3 de Aminoácido Excitatório/metabolismo , Feminino , Células HEK293 , Humanos , Imuno-Histoquímica , Hibridização In Situ , Rim/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Proteolipídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta/genéticaRESUMO
Among amino acids, leucine is a potential signaling molecule to regulate cell growth and metabolism by activating mechanistic target of rapamycin complex 1 (mTORC1). To reveal the critical structures of leucine molecule to activate mTORC1, we examined the structure-activity relationships of leucine derivatives in HeLa S3 cells for cellular uptake and for the induction of phosphorylation of p70 ribosomal S6 kinase 1 (p70S6K), a downstream effector of mTORC1. The activation of mTORC1 by leucine and its derivatives was the consequence of two successive events: the cellular uptake by L-type amino acid transporter 1 (LAT1) responsible for leucine uptake in HeLa S3 cells and the activation of mTORC1 following the transport. The structural requirement for the recognition by LAT1 was to have carbonyl oxygen, alkoxy oxygen of carboxyl group, amino group and hydrophobic side chain. In contrast, the requirement for mTORC1 activation was more rigorous. It additionally required fixed distance between carbonyl oxygen and alkoxy oxygen of carboxyl group, and amino group positioned at α-carbon. L-Configuration in chirality and appropriate length of side chain with a terminal isopropyl group were also important. This confirmed that LAT1 itself is not a leucine sensor. Some specialized leucine sensing mechanism with rigorous requirement for agonistic structures should exist inside the cells because leucine derivatives not transported by LAT1 did not activate mTORC1. Because LAT1-mTOR axis is involved in the regulation of cell growth and cancer progression, the results from this study may provide a new insight into therapeutics targeting both LAT1 and leucine sensor.
Assuntos
Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Leucina/farmacologia , Complexos Multiproteicos/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Transporte Biológico , Expressão Gênica , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Transportador 1 de Aminoácidos Neutros Grandes/genética , Leucina/análogos & derivados , Leucina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/genética , Fosforilação/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Transdução de Sinais , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/genéticaRESUMO
3-(18)F-l-α-methyl-tyrosine ([18F]FAMT), a PET probe for tumor imaging, has advantages of high cancer-specificity and lower physiologic background. FAMT-PET has been proved useful in clinical studies for the prediction of prognosis, the assessment of therapy response and the differentiation of malignant tumors from inflammation and benign lesions. The tumor uptake of [18F]FAMT in PET is strongly correlated with the expression of L-type amino acid transporter 1 (LAT1), an isoform of system L upregulated in cancers. In this study, to assess the transporter-mediated mechanisms in FAMT uptake by tumors, we examined amino acid transporters for FAMT transport. We synthesized [14C]FAMT and measured its transport by human amino acid transporters expressed in Xenopus oocytes. The transport of FAMT was compared with that of l-methionine, a well-studied amino acid PET probe. The significance of LAT1 in FAMT uptake by tumor cells was confirmed by siRNA knockdown. Among amino acid transporters, [14C]FAMT was specifically transported by LAT1, whereas l-[14C]methionine was taken up by most of the transporters. Km of LAT1-mediated [14C]FAMT transport was 72.7 µM, similar to that for endogenous substrates. Knockdown of LAT1 resulted in the marked reduction of [14C]FAMT transport in HeLa S3 cells, confirming the contribution of LAT1 in FAMT uptake by tumor cells. FAMT is highly specific to cancer-type amino acid transporter LAT1, which explains the cancer-specific accumulation of [18F]FAMT in PET. This, vice versa, further supports the cancer-specific expression of LAT1. This study has established FAMT as a LAT1-specific molecular probe to monitor the expression of a potential tumor biomarker LAT1.
Assuntos
Biomarcadores Tumorais/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/fisiologia , Metiltirosinas/metabolismo , Compostos Radiofarmacêuticos/metabolismo , Animais , Transporte Biológico , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , RNA Interferente Pequeno/genética , Xenopus laevisRESUMO
A photo-sensitive amino acid analogue was introduced into an outer membrane lipoprotein, Pal, and then subjected to photo-crosslinking with the lipoprotein-specific ABC transporter LolCDE. Pal crosslinked to LolE but not LolC in vivo despite that both are structurally similar membrane subunits. LolCDE liganded with Pal containing the photo-sensitive amino acid analogue was isolated and subjected to in vitro photo-crosslinking. LolE was found to be the binding site for Pal. ATP binding to LolD decreased the LolE-Pal crosslinking by decreasing their hydrophobic interaction. ATP hydrolysis in the presence of LolA completely abolished the LolE-Pal crosslinking and, concomitantly, generated a new LolA-Pal crosslinked product.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Complexos Multiproteicos/metabolismo , Subunidades Proteicas/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Trifosfato de Adenosina/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Benzofenonas/química , Western Blotting , Cromatografia de Afinidade , Códon de Terminação , Reagentes de Ligações Cruzadas/química , Eletroforese em Gel de Poliacrilamida , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/isolamento & purificação , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/química , Processos Fotoquímicos , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Millions of molecules of lipopolysaccharide (LPS) must be assembled on the Escherichia coli cell surface each time the cell divides. The biogenesis of LPS requires seven essential lipopolysaccharide transport (Lpt) proteins to move LPS from the inner membrane through the periplasm to the cell surface. However, no intermediate transport states have been observed. We developed methods to observe intermediate LPS molecules bound to Lpt proteins in the process of being transported in vivo. Movement of individual LPS molecules along these binding sites required multiple rounds of adenosine triphosphate (ATP) hydrolysis in vitro, which suggests that ATP is used to push a continuous stream of LPS through a transenvelope bridge in discrete steps against a concentration gradient.